Adhesive for medical applications and means for haemostasis

ABSTRACT

The invention relates to adhesives for medical applications and methods of their preparation and use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation, of U.S. application Ser. No.12/223,772, filed on Dec. 22, 2008 now U.S. Pat. No. 7,923,003, saidapplication is a national phase entry under 35 U.S.C. §371 ofInternational Application No. PCT/EP2007/001131 filed Feb. 9, 2007,which claims priority from German Application No. 10 2006 006 904.8filed Feb. 9, 2006, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to adhesives for medical applications and meansfor haemostasis. In particular, the invention relates to a kit for themanufacture of an adhesive suitable for adhering hard and soft tissue toeach other, for the closure of wounds, as well as the adhesive itselfand its uses in the medical field. The kit and the correspondingadhesive comprise, as individual components, substrates of thepolyphenoloxidase as well as polyphenoloxidases as such. Furthermore,the invention relates to novel haemostatics as well as the combined useof haemostatics and adhesives in the closure of wounds.

The development of novel adhesives for tissue that can be applied,especially in problematic cases, has strongly gained importance in thepast few years. Adhesives for tissue on the basis of acrylate used sofar can only be applied to superficial wounds, since acrylates are notdecomposed biologically and could impose a toxic hazard. One focus is,therefore, on the biological decomposability of the used materials.Therefore, polymers of vegetable or animal origin are of specialimportance in the manufacture of adhesives. Mussels can have strongbonds with various carrier substrates under water. The reason for thisis the formation/excretion of MAPs (mussel adhesive proteins). MAPsconsist of a number of proteins that can vary depending on the type ofmussel. In U.S. Pat. No. 5,015,677 A, a mussel adhesive is protectedcontaining as an essential component a very special decapeptide isolatedfrom mussels. This decapeptide contains dehydroxyaromates assubstituents of very special amino acids that form an essentialcomponent of the peptide chain. The peptide chains are then bonded witheach other by means of a tyrosinase. Further satisfying solutions thatmake it possible to bond bones with implants by means of an adhesive arenot known.

EP 0 947 142 discloses increasing the molecular weight of proteins bycrosslinking by means of multi-copper enzymes. The described increasingof the molecular weight of the substrate proteins occurred seventeenhours after incubation. The proteins crosslinked using the method of EP0 947 142 are especially suitable for the use in food, for example formodifying the consistence of sausage. Crosslinking of proteins by meansof substituted dihydroxyaromates is not disclosed in EP 0 947 142.

If adhesives for wounds are intended to be used in case of stronglybleeding wounds, there is the danger that adhesive components are washedaway with the bloodstream and the adhesion occurs at a site where it isnot desired. Thus, the danger of thrombosis is strongly increased. Acutetreatment of strongly bleeding wounds is still a problem that is notsolved satisfyingly. Haemostasis according to the state of the art canbe achieved by

(a) Denaturation of proteins, for example by means of metal salts

(b) Microporous biopolymers or biopolymers having a great inner surface,respectively. They resorb water, and, therefore, cause concentration ofblood cells as well as coagulation factors. Polysaccharides consistingof poly-N-acetyl-glucosamines or chitosan have proved to be especiallyadvantageous. They are, for example, contained in the skeleton ofinsects, but also in algae. Biopolymers such as gelatin, collagen,fibrin sponges or oxidized cellulose have a great inner surface. Aconcentration of coagulation factors is caused by absorption onto thesegreat surfaces.

(c) Enzymes, especially by thrombin or proteolytic enzymes havingthrombin-like activity with or without the addition of calcium.

(d) Highly concentrated coagulation factors

(e) Combinations of (c) and (d)

(f) Calcium alginates

(g) Alumosilicate synthesis products having a microporous structure.

Alumosilicate synthesis products used for haemostasis are alkaline oralkaline-earth alumosilicates of differing composition.

The company Z-Medica, U.S. has developed a synthesis product on thebasis of synthesized earth-alkali alumosilicates that completelydehydrate at above 400° C. for the immediate treatment of war injuriesand for emergency use. Because of the strongly exothermic reaction,blood is dehydrated causing haemostasis. The synthesis product isdirectly applied to the wound as a granulate. It is marketed under thedesignation “Quikclot.” A disadvantage has been described for thisproduct in that because of the exothermic reaction (temperatures up to60° C.) adjacent tissue can be damaged (Journal of Trauma 54 (2003) 6,1077-1082).

Synthetic lithium alumosilicates (EP 1 176 991 A1, WO 00/69480) weredeveloped especially for the treatment of wounds and are marketed underthe designation CERDAC. In the case of CERDAC, a microporous ceramicproduct is produced using high temperatures (>1000° C.) in order toobtain optimal treatment of wounds.

The capillary force action is too low, apart from the partly complicatedproduction, to satisfy all requirements.

A great problem is still the possible chronification of wounds,especially in case of risk patients. This so far cannot be avoided forcertain, and the treatment of such wounds is still very difficult.Chronification often involves an infection of the wound.

Four million patients suffer from chronically open wounds in Germanyalone. The annual costs amount to E1.7 to £3.2 billion. The treatment ofchronic wounds so far has not been solved satisfyingly.

BRIEF SUMMARY OF THE INVENTION

It can be summarized that for the problem of adhering hard and softtissue as well as for the rapid closure, the treatment of stronglybleeding and especially the treatment of chronic wounds, no completelysatisfying solutions have been achieved yet.

The present object is to solve the problem described in the prior art bymeans of products having efficient adhesive properties for hard and softtissue. In particular, systems such as adhesives should be provided,enabling stopping the bleeding of strongly bleeding wounds and/orstrongly adhering bone parts with each other or bone parts withimplants. Further desired are adhesives enabling soft tissue with bones,such as cartilage transplants. The inventive possibilities for theclosure of wounds should preferably be usable for chronic and infectedwounds.

The finding of the present invention is that polymers that have at leastone free amino group are crosslinked by means of bridge molecules using(lignolytic) polyphenoloxidases to provide covalent linkages with eachother and/or with body tissue so that they can be used as an adhesivefor hard and soft tissue.

A further finding of the present invention is that specificallyprocessed natural zeolites cause a rapid stopping of bleeding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing cell adhesion on uncoated and collagen coatedsurfaces.

DETAILED DESCRIPTION

In this respect, the present invention relates in a first embodiment toa kit, comprising the following individual components:

(a) Polymers having at least one free amino group,

(b) Bridge molecules, selected from the group consisting of monocyclicortho-dihydroxyaromates, monocyclic para-dihydroxyaromates, bicyclicmonohydroxyaromates, polycyclic monohydroxyaromates, bicyclicdihydroxyaromates, polycyclic dihydroxyaromates, bicyclictrihydroxyaromates, polycyclic trihydroxyaromates, and mixtures thereof,and

(c) Polyphenoloxidases, in particular lignolytic polyphenoloxidases,

wherein both individual components (components (b) and (c)), i.e. thebridge molecules polyphenoloxidases, in particular lignolyticpolyphenoloxidases, are not in contact.

In the context of the present invention, a kit is particularly a packageproviding for the storage of the individual components next to eachother, so that they are not in contact. In the present case, at leastthe bridge molecules (component (b)) need to be separated from the(lignolytic) polyphenoloxidases (component (c)), in order to avoid apremature and undesired reaction. Thus, a mixture of components (a) and(b) and/or components (a) and (c) is possible.

Both individual components may preferably be stored in differentcontainers or chambers, in order to avoid contact between the bridgemolecules and the (lignolytic) polyphenoloxidases. This is also true incase that one or both individual components (b) and (c) are mixed withthe polymer (a). A syringe may be considered enabling the administrationof both individual components, i.e. the bridge molecules and the(lignolytic) polyphenoloxidases, separate from each other by means oftwo cannulae. Here it is preferred that both individual components, i.e.the bridge molecules and the (lignolytic) polyphenoloxidases, arepresent in the syringe in different chambers. Of course, it may also beconsidered that the kit comprises two syringes wherein one contains thebridge molecules and the other the (lignolytic) polyphenoloxidases.

In one embodiment, the kit is a double-chambered syringe, preferablyalready having a mixing extruder added. This provides for an especiallyexact dosing and ease of handling. A double-chambered syringe of theMixpac type (Mixpac Systems AG, Rotkreuz, Switzerland) is, for example,preferred.

It is to be understood that in the context of the invention, one or moredifferent, individual components (a), (b), and (c) may independently beused. Singular and plural is, therefore, used for all componentssynonymously.

It is an essential requirement that the kit comprises polymers (a)having at least one free amino group as an individual component. In thepresent invention, a free amino group means an amino group that is notpart of the polymer chain, but is a radical of the polymer chain. Thesepolymers are not part of the human body, since they are present in thekit. The polymers are preferably polymers, having more than one aminogroup, i.e. polymers rich in amino groups, such as peptides.

Peptides (i.e. oligopeptides and proteins) naturally have an amide bondbetween the amino acids. The peptides preferably contain lysine. In apreferred embodiment of the invention, lysine-containing oligopeptidesare used. Collagen may be considered as the peptide.

If the polymer is a peptide, there is a special advantage in that thecompound obtained by reaction of the component is biologicallydecomposable. By linking peptide sequences containing the amino acidsrelevant for the adhesive action, a very strong adhesion is formed thatcan, however, be resorbed during the process of healing. The initialhardening phase involving highly adhesive properties, and thetime-depending slow absorption into the body during advanced healingprocesses are especially advantageous, since body tissue is increasinglyfixed into the adhesion site.

In the present invention, an oligopeptide (2 to about 100 amino acidslong, preferably about 4 to about 20 amino acids long, or about 6 toabout 10 amino acids long) as well as a protein (about 100 to about5,000 amino acids long, preferably 100-1,000 or 100-200 amino acidslong) is considered a peptide. The peptide is preferably 10-1,000 aminoacids long. The peptide can have a molecular weight of about 1 to about100 or to about 200 kDa, in particular about 2 to about 50 kDa, or about5 to about 20 kDa. It can be modified or substituted, e.g. can beglycosylized. Apart from conventional proteinogenic amino acids,modified or atypical amino acids such as hydroxylysine may be containedin the oligopeptide. The use of D-amino acids in place of or in additionto L-amino acids is possible and retards decomposition of the peptide.

The amino groups may preferably be primary or secondary amino groups.However, primary amino groups are especially reactive. At least one ofthe reactive groups of the peptide is part of a diamino acid, e.g.lysine. Therefore, the peptide preferably comprises at least one diaminoacid, preferably at least 2, 3, 4, 5 or more diamino acids. As mentionedabove, lysine-containing peptides (oligopeptides or proteins) arepreferably used.

Other amino acids, such as arginine, asparagine, glutamine, or histidinealso have reactive amino groups that may react with thedihydroxyaromates.

The amino groups, especially the amino groups provided by the diaminoacid, are especially suitable for the crosslinking reaction between thepeptide and the bridge molecule. However, hydroxyl groups and mercaptogroups in the peptide may also contribute to the crosslinking reaction.Therefore, the peptide preferably comprises at least one amino acidhaving a hydroxyl group, which means especially serine, threonine, ortyrosine, or a mercapto group, e.g. cysteine. Hydroxylysine orpolyphenolic amino acid components, of the type present in MAPs, may bepresent in the peptides used according to the invention. An advantage ofthe invention is, however, that the presence of these specific aminoacid components and, therefore, the use of MAPs is not mandatory.Therefore, in a preferred embodiment, the polymer is free ofpolyphenolic amino acid components, i.e. is free of MAPs. The usedpeptides may easily be prepared using recombination.

The crosslinking obtained by reaction of the bridge molecules with apeptide also depends on the proportion of the available reactive groupsin the peptide. Good adhesive properties may be obtained with an amountof amino acids of the peptide having a reactive amino group (e.g.diamino acids such as lysine) of at least 10%. If the polymer is not apeptide the amount of structural elements of the polymer having an aminogroup is preferably at least 10%. However, the amount of these aminoacids or structural elements is more preferably higher, at least 20%, atleast 30%, at least 40%, or at least 50%, or even at least 80% to 100%.Naturally occurring peptides and proteins, e.g. albumine or caseine, maybe used, MAPs are especially suitable.

However, shorter peptides that can easily be prepared synthetically mayfurther also be used. In an especially preferred embodiment of theinvention, at least 50% of the amino acids of the peptide is lysine.Lysine and a further amino acid may, for example, by arranged as arepeating dipeptide unit. However, a different sequence or theincorporation of further amino acids, in particular of arginine,asparagine, glutamine, or histidine (in place of or in addition tolysine), serine, or threonine (in place of or in addition to tyrosine),of cysteine or other amino acids is possible. The peptide lengths ofabout 10-20 amino acids or a mixture of peptides having different chainlengths is especially preferred.

In a preferred embodiment, the polymers consist exclusively of two aminoacids, for example (lysine-thyrosine)_(n), wherein n can have valuesbetween 5 and 40, such as 5, 10, or 20.

A further essential requirement of the present invention is that the kitcomprises bridge molecules causing a crosslinking of polymers that haveat least one free amino group (such as diamino acids).

Generally, substituted dihydroxyaromates and/or substrates of the(lignolytic) polyphenoloxidases, such as laccases, are suitable asbridge molecules.

Thus, monocyclic ortho-dihydroxyaromates, monocyclicpara-dihydroxyaromates, bicyclic monohydroxyaromates, polycyclicmonohydroxyaromates, bicyclic dihydroxyaromates, polycyclicdihydroxyaromates, bicyclic trihydroxyaromates, polycyclictrihydroxyaromates, or mixtures thereof are used. The hydroxyaromatesaccording to the invention are not part of the polymer chain, as opposedto the known mussel adhesives.

It may be considered that these aromates are further substituted.Preferred functional groups are substituents selected from the groupconsisting of halogen, sulfo-, sulfon-, sulfamido, sulfanyl-, amino-,amido-, azo-, immino-, and hydroxyl. Here it can especially be foundthat substituted aromates, in particular substituted dihydroxyaromateshave surprisingly advantageous polymerization properties, in particulara rapid polymerization, a low self-coupling, and a good steadiness ofthe adhesion is obtained. Suitable substitution of the aromates maycause that even monohydroxyaromates as bridge molecules are suitable forcrosslinking. Substituted in the context of this invention means, inparticular, that 1, 2, 3, or 4 further radicals are bonded to thearomates apart from the hydroxyl groups. On the other hand,monohydroxylated biaryl compounds are also suitable.

Phenol derivatives having a hydroxyl group or a methoxy group in ortho-or para position are especially preferred. Thus, the following compoundsof formulae 3 and 4 are preferred:

wherein

n=0-10, preferably 0 or 1, especially 0,

R₁═OH or NH₂ or Hal, preferably OH, Cl, or Br, especially OH,

R₂═H, CH₃, CHO, COCH₃, CONH₂, CON-alkyl, CON-alkyl-OH, COOH, COO-alkyl,alkyl, substituted aromate, especially CON-alkyl or COO-alkyl, and

R₃═H, CH₃, alkyl, substituted aromate, especially H or CH₃.

Alkyl means branched or non-branched aliphatic hydrocarbon chains,preferably having 1-20, more preferably 1-6 carbon atoms, e.g. methyl,ethyl, propyl, butyl, isobutyl, n-pentyl, n-hexyl.

Compounds of formula 3 and the hydrochinone thereof, which may befurther substituted, are possible. In view of a rapid adhesive reaction,substituted dihydroxyaromates having a low self-coupling are especiallysuitable according to the invention. 2,5-dihydroxybenzamide ispreferably used, 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide isespecially preferred.

In the case of trihydroxyaromates, it is preferred that not more thantwo hydroxy groups are present per benzene unit. Especially preferredare polyphenyls, i.e. biphenyl or triphenyl of the following formula 5:

wherein

n=0-10, preferably 0 or 1

and

R₁═H and R₂═OH or R₁═OH and R₂═H.

The phenyls of formula 5 may be substituted, e.g. in ortho position ofthe OH group with CH₃, CHO, COCH₃, CONH₂, CON-alkyl, CON-alkyl-OH, COOH,COO-alkyl, alkyl, substituted aromate, especially CON-alkyl orCOO-alkyl, and/or in meta position of the OH group with CH₃, alkyl,substituted aromate, especially CH₃.

It is further required that the kit comprises polyphenoloxidases as afurther individual component, such as lignolytic polyphenoloxidases,especially laccases (EC 1.10.3.2). Laccases are known in the art. Theymay be of plant, fungi, bacteria, or insect origin, or be derived fromnatural enzymes. Laccases used according to the invention may beprepared or purified using recombination. Generally, special purity ofthe laccase is not required, fluids of lignolytic fingi may optionallybe used. However, for medical applications, substantial removal ofmicrobiological substances, such as lipopolysaccharides or othercomponents of the cell wall, is often desirable. Examples are laccase ofthe genus Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes,Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia,Coprinus, Psatyrella, Myceliophthora, Schtalidium, Polyporus, Phlebia,or Coriolus. The manufacture of laccases is disclosed, for example, inEP 0 947 142.

By the use of polyphenoloxidases, such as lignolytic polyphenoloxidases,preferably laccase (EC 1.10.3.2) in the kit, their broad as well asspecial substrate spectrum can be used for the adhesive reaction.Therefore, the kit is especially characterized in that the adhesivereaction is not limited to special natural peptides acting as bridgemolecules, but a broad spectrum of bridge molecules, on the one hand,and of peptides such as oligopeptides or proteins, on the other hand,may be used.

Variations of the amounts of the individual component (a), in particularof the peptide, as well as of the bridge molecule, are possible in therange of about 1-50 mM. For each application the optimal ratio of theamounts is to be determined using preliminary tests.

It has to be considered that depending on the selected bridge molecule,a reaction of the bridge molecule with itself takes place decreasing theformation of crosslinking products. Too low a concentration of thebridge molecules leads to a slow reaction, too high a concentration toincreased by reactions due to self-coupling. When a lower ratio ofpeptides is present, reactive amino-, mercapto and/or hydroxyl groups ofthe substrates to be adhered are increasingly included in thecrosslinking. The amount of polyphenoloxidase has an influence on thereaction rate wherein the rapid reach of the gel point or the completehardening or prolonged processability of the combination can be obtaineddepending on the application. The kit can be adjusted to the respectiveconcrete problem by optimizing the ratio of amounts in preliminarytests.

It is a special advantage of the kit that the components in the kit arepresent in an optimized ratio of amounts appropriate for the intendeduse. A preferred ratio of amounts for adhering soft tissue is, forexample, [Tyr-Lys]_(n), n=4-35, 8.5 mM;2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide 12.5 mM; polyphenoloxidase:32 U (156 nmol ml⁻¹ min⁻¹). The ratio of amounts may be selected inadvance in the kit and prevents complicated individual dosing of theingredients.

The combination used in the kit can contain further additives andadjuvants, e.g. fillers, such as collagen, albumine, hyaluronic acid orthe like. The total proportion of individual component (a), such aspeptide, and bridge molecule is preferably about 50%-90%.

The essential components of the kit are preferably dissolved in one ormore aqueous solvents. For medical applications, the solvent isnon-toxic and biologically acceptable. A phosphate buffer, such ascalcium phosphate or sodium phosphate buffer or PBS, is preferred as asolvent for biological applications. For other applications, an organicsolvent, such as DMSO, or a mixture of an aqueous and an organicsolvent, may be used. Alternatively, the combination or its componentsmay be dissolved in the solvent and introduced into the kit only priorto use.

The consistency of the components used in the kit does not, however,need to be liquid, but may have a paste-like consistency. The viscosityand flowability of the used individual components may be adjusted,depending on e.g. the length of the wound to be fixed and the depth ofthe wound gap or the substrates to be adhered, respectively. Apart fromthe length of the used peptides/polymers, the amount of solvent also hasan influence on these parameters. Additives, such as thixotropic agents,may be used for adjusting viscosity and flowability. Typically, acombination of higher viscosity is used for adhering hard tissue thanfor adhering soft tissue.

The pH is preferably 2-10, especially 5-7. The reaction can proceed at2-80° C.; however, the temperature is preferably about 20-37° C. or25-30° C. Thus, the adhesive reaction of the kit can be carried out atroom temperature/body temperature.

There are no special requirements for storage, unlike in the case offrozen fibrin adhesives. The (lignolytic) polyphenoloxidase, such aslaccase, may be used in dissolved form, wherein storage at temperaturesof a refrigerator is sufficient. It is also possible to provide thelignolytic polyphenoloxidase, such as laccase, in the form of a powderand to dissolve it in advance of an intended application.

Sterilization of the kit or its individual components may preferably beachieved without structural changes. For example, a solution may besterilized using filtration. However, sterilization by means of gammaradiation is preferred, since this can be carried out after packaging,so that packaging under aseptic conditions is unnecessary. The loss ofactivity of the (lignolytic) polyphenoloxidase, such as laccase, due togamma sterilization which can amount to up to 50% may be compensatedusing a correspondingly higher initial concentration.

The above-defined kit is preferably used as a medicament. Classificationas a pharmaceutical preparation or a medical preparation is alsopossible depending on national law. These terms can be interchanged inthe context of the invention.

In a further aspect, the present invention relates to an adhesive havingthe individual components described above.

Thus, the adhesive comprises the following individual components:

(a) Polymers having at least one free amino group, wherein the polymersare not part of the human body,

(b) Bridge molecules, selected from the group consisting of monocyclicortho-dihydroxyaromates, monocyclic para-dihydroxyaromates, bicyclicmonohydroxyaromates, polycyclic monohydroxyaromates, bicyclicdihydroxyaromates, polycyclic dihydroxyaromates, bicyclictrihydroxyaromates, polycyclic trihydroxyaromates, and mixtures thereof,and

(c) Polyphenoloxidases, especially lignolytic polyphenoloxidases,

wherein both individual components (components (b) and (c)), i.e. thebridge molecules with the polyphenoloxidases, especially the lignolyticpolyphenoloxidases, are not in contact, i.e. mixed with each other.

As regards the individual components, it is referred to the descriptionof the kit.

It is, therefore, to be understood that, according to the invention, oneor more different individual components (a), (b), and (c) may be usedindependently of each other. Singular and plural is, therefore, usedsynonymously with regard to all components.

It is an essential requirement that the adhesive comprises polymers (a)having at least one free amino group as an individual component. In thepresent invention, a free amino group means an amino group that is notpart of the polymer chain, but is a radical of the polymer chain. Thesepolymers are not part of the human body, since they are present in theadhesive. The polymers are preferably polymers having more than oneamino group, i.e. polymers rich in amino groups, such as peptides.

Peptides (i.e. oligopeptides and proteins) naturally have an amide bondbetween the amino acids. The peptides preferably contain lysine. In apreferred embodiment of the invention, lysine-containing oligopeptidesare used. Collagen may be considered as the peptide.

If the polymer is a peptide, there is a special advantage in that thecompound obtained by reaction of the component is biologicallydecomposable. By linking peptide sequences containing the amino acidsrelevant for the adhesive action, a very strong adhesion is formed thatcan, however, be resorbed during the process of healing. The initialhardening phase involving highly adhesive properties, and thetime-depending slow absorption into the body during advanced healingprocesses are especially advantageous, since body tissue is increasinglyfixed into the adhesion site.

In the present invention, an oligopeptide (2 to about 100 amino acidslong, preferably about 4 to about 20 amino acids long, or about 6 toabout 10 amino acids long) as well as a protein (about 100 to about5,000 amino acids long, preferably 100-1,000 or 100-200 amino acidslong) is considered a peptide. The peptide is preferably 10-1,000 aminoacids long. The peptide can have a molecular weight of about 1 to about100 or to about 200 kDa, in particular about 2 to about 50 kDa, or about5 to about 20 kDa. It can be modified or substituted, e.g. can beglycosylized. Apart from conventional proteinogenic amino acids,modified or atypical amino acids such as hydroxylysine may be containedin the oligopeptide. The use of D-amino acids in place of or in additionto L-amino acids is possible and retards decomposition of the peptide.

The amino groups may preferably be primary or secondary amino groups.However, primary amino groups are especially reactive. At least one ofthe reactive groups of the peptide is part of a diamino acid, e.g.lysine. Therefore, the peptide preferably comprises at least one diaminoacid, preferably at least 2, 3, 4, 5 or more diamino acids. As mentionedabove, lysine-containing peptides (oligopeptides or proteins) arepreferably used.

Other amino acids, such as arginine, asparagine, glutamine, or histidinealso have reactive amino groups that may react with thedihydroxyaromates.

The amino groups, especially the amino groups provided by the diaminoacid, are especially suitable for the crosslinking reaction between thepeptide and the bridge molecule. However, hydroxyl groups and mercaptogroups in the peptide may also contribute to the crosslinking reaction.Therefore, the peptide preferably comprises at least one amino acidhaving a hydroxyl group, which means especially serine, threonine, ortyrosine, or a mercapto group, e.g. cysteine. Hydroxylysine orpolyphenolic amino acid components, of the type present in MAPs, may bepresent in the peptides used according to the invention. An advantage ofthe invention is, however, that the presence of these specific aminoacid components and, therefore, the use of MAPs is not essentiallyrequired. Therefore, in a preferred embodiment, the polymer is free ofpolyphenolic amino acid components, i.e. is free of MAPs. The usedpeptides may easily be prepared using recombination.

The crosslinking obtained by reaction of the bridge molecules with apeptide also depends on the proportion of the available reactive groupsin the peptide. Good adhesive properties may be obtained with an amountof amino acids of the peptide having a reactive amino group (e.g.diamino acids such as lysine) of at least 10%. If the polymer is not apeptide the amount of structural elements of the polymer having an aminogroup is preferably at least 10%. However, the amount of these aminoacids or structural elements is more preferably higher, at least 20%, atleast 30%, at least 40%, or at least 50%, or even at least 80% to 100%.Naturally occurring peptides and proteins, e.g. albumine or caseine, maybe used, MAPs are especially suitable.

However, shorter peptides that can easily be prepared synthetically mayfurther also be used. In an especially preferred embodiment of theinvention, at least 50% of the amino acids of the peptide is lysine.Lysine and a further amino acid may, for example, by arranged as arepeating dipeptide unit. However, a different sequence or theincorporation of further amino acids, in particular of arginine,asparagine, glutamine, or histidine (in place of or in addition tolysine), serine, or threonine (in place of or in addition to tyrosine),of cysteine or other amino acids is possible. The peptide lengths ofabout 10-20 amino acids or a mixture of peptides having different chainlengths is especially preferred.

In a preferred embodiment, the polymers consist exclusively of two aminoacids, for example (lysine-thyrosine), wherein n can have values between5 and 40, such as 5, 10, or 20.

A further essential requirement of the present invention is that theadhesive comprises bridge molecules causing a crosslinking of polymersthat have at least one free amino group (such as diamino acids), asdefined above.

Generally, substituted dihydroxyaromates and/or substrates of the(lignolytic) polyphenoloxidases, such as laccases, are suitable asbridge molecules.

Thus, monocyclic ortho-dihydroxyaromates, monocyclicpara-dihydroxyaromates, bicyclic monohydroxyaromates, polycyclicmonohydroxyaromates, bicyclic dihydroxyaromates, polycyclicdihydroxyaromates, bicyclic trihydroxyaromates, polycyclictrihydroxyaromates, or mixtures thereof are used. The hydroxyaromatesaccording to the invention are not part of the polymer chain, as opposedto the known mussel adhesives.

It may be considered that these aromates are further substituted.Preferred functional groups are substituents selected from the groupconsisting of halogen, sulfa-, sulfon-, sulfamido, sulfanyl-, amino-,amido-, azo-, immino-, and hydroxyl. Here it can especially be foundthat substituted aromates, in particular substituted dihydroxyaromateshave surprisingly advantageous polymerization properties, in particulara rapid polymerization, a low self-coupling, and a good steadiness ofthe adhesion is obtained. Suitable substitution of the aromates maycause that even monohydroxyaromates as bridge molecules are suitable forcrosslinking. Substituted in the context of this invention means, inparticular, that 1, 2, 3, or 4 further radicals are bonded to thearomates apart from the hydroxyl groups. On the other hand,monohydroxylated biaryl compounds are also suitable.

Phenol derivatives having a hydroxyl group or a methoxy group in ortho-or para position are especially preferred. Thus, the following compoundsof formulae 3 and 4 are preferred:

wherein

n=0-10, preferably 0 or 1, especially 0,

R₁═OH or NH₂ or Hal, preferably OH, Cl, or Br, especially OH,

R₂═H, CH₃, CHO, COCH₃, CONH₂, CON-alkyl, CON-alkyl-OH, COOH, COO-alkyl,alkyl, substituted aromate, especially CON-alkyl or COO-alkyl, and

R₃═H, CH₃, alkyl, substituted aromate, especially H or CH₃.

Alkyl means branched or non-branched aliphatic hydrocarbon chains,preferably having 1-20, more preferably 1-6 carbon atoms, e.g. methyl,ethyl, propyl, butyl, isobutyl, n-pentyl, n-hexyl.

Compounds of formula 3 and the hydrochinone thereof, which may befurther substituted, are possible. In view of a rapid adhesive reaction,substituted dihydroxyaromates having a low self-coupling are especiallysuitable according to the invention. 2,5-dihydroxybenzamide ispreferably used, 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide isespecially preferred.

In the case of trihydroxyaromates, it is preferred that not more thantwo hydroxy groups are present per benzene unit. Especially preferredare polyphenyls, i.e. biphenyl or triphenyl of the following formula 5:

wherein

n=0-10, preferably 0 or 1

and

R₁═H and R₂═OH or R₁═OH and R₂═H.

The phenyls of formula 5 may be substituted, e.g. in ortho position ofthe OH group with CH₃, CHO, COCH₃, CONH₂, CON-alkyl, CON-alkyl-OH, COOH,COO-alkyl, alkyl, substituted aromate, especially CON-alkyl orCOO-alkyl, and/or in meta position of the OH group with CH₃, alkyl,substituted aromate, especially CH₃.

It is further required that the adhesive comprises polyphenoloxidases asa further individual component, such as lignolytic polyphenoloxidases,especially laccases (EC 1.10.3.2). Laccases are known in the art. Theymay be of plant, fungi, bacteria, or insect origin, or be derived fromnatural enzymes. Laccases used according to the invention may beprepared or purified using recombination. Generally, special purity ofthe laccase is not required, fluids of lignolytic fingi may optionallybe used. However, for medical applications, substantial removal ofmicrobiological substances, such as lipopolysaccharides or othercomponents of the cell wall, is often desirable. Examples are laccase ofthe genus Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes,Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia,Coprinus, Psatyrella, Myceliophthora, Schtalidium, Polyporus, Phlebia,or Coriolus. The manufacture of laccases is disclosed, for example, inEP 0 947 142.

By the use of polyphenoloxidases, such as lignolytic polyphenoloxidases,preferably laccase (EC 1.10.3.2) in the adhesive, their broad as well asspecial substrate spectrum can be used for the adhesive reaction.Therefore, the adhesive is especially characterized in that the adhesivereaction is not limited to special natural peptides acting as bridgemolecules, but a broad spectrum of bridge molecules, on the one hand,and of peptides such as oligopeptides or proteins, on the other hand,may be used.

Variations of the amounts of the individual component (a), in particularof the peptide, as well as of the bridge molecule, are possible in therange of about 1-50 mM. For each application the optimal ratio of theamounts is to be determined using preliminary tests.

It has to be considered that depending on the selected bridge molecule,a reaction of the bridge molecule with itself takes place decreasing theformation of crosslinking products. Too low a concentration of thebridge molecules leads to a slow reaction, too high a concentration toincreased by reactions due to self-coupling. When a lower ratio ofpeptides is present, reactive amino-, mercapto and/or hydroxyl groups ofthe substrates to be adhered are increasingly included in thecrosslinking. The amount of polyphenoloxidase has an influence on thereaction rate wherein the rapid reach of the gel point or the completehardening or prolonged processability of the combination can be obtaineddepending on the application. The adhesive can be adjusted to therespective concrete problem by optimizing the ratio of amounts inpreliminary tests.

It is a special advantage of the adhesive that the components in theadhesive are present in an optimized ratio of amounts appropriate forthe intended use. A preferred ratio of amounts for adhering soft tissueis, for example, [Tyr-Lys]_(n), n=4-35, 8.5 mM;2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide 12.5 mM; polyphenoloxidase:32 U (156 nmol ml⁻¹ min⁻¹). The ratio of amounts may be selected inadvance in the adhesive and prevents complicated individual dosing ofthe ingredients.

The combination used in the adhesive can contain further additives andadjuvants, e.g. fillers, such as collagen, albumine, hyaluronic acid orthe like. The total proportion of individual component (a), such aspeptide, and bridge molecule is preferably about 50%-90%.

The essential components of the adhesive are preferably dissolved in oneor more aqueous solvents. For medical applications, the solvent isnon-toxic and biologically acceptable. A phosphate buffer, such ascalcium phosphate or sodium phosphate buffer or PBS, is preferred as asolvent for biological applications. For other applications, an organicsolvent, such as DMSO, or a mixture of an aqueous and an organicsolvent, may be used. Alternatively, the combination or its componentsmay be dissolved in the solvent and introduced into the adhesive onlyprior to use.

The consistency of the components used in the adhesive does not,however, need to be liquid, but may have a paste-like consistency. Theviscosity and flowability of the used individual components may beadjusted, depending on e.g. the length of the wound to be fixed and thedepth of the wound gap or the substrates to be adhered, respectively.Apart from the length of the used peptides/polymers, the amount ofsolvent also has an influence on these parameters. Additives, such asthixotropic agents, may be used for adjusting viscosity and flowability.Typically, a combination of higher viscosity is used for adhering hardtissue than for adhering soft tissue.

The pH is preferably 2-10, especially 5-7. The reaction can proceed at2-80° C.; however, the temperature is preferably about 20-37° C. or25-30° C. Thus, the adhesive reaction of the adhesive can be carried outat room temperature/body temperature.

There are no special requirements for storage, unlike in the case offrozen fibrin adhesives. The (lignolytic) polyphenoloxidase, such aslaccase, may be used in dissolved form, wherein storage at temperaturesof a refrigerator is sufficient. It is also possible to provide thelignolytic polyphenoloxidase, such as laccase, in the form of a powderand to dissolve it in advance of an intended application.

Sterilization of the adhesive or its individual components maypreferably be achieved without structural changes. For example, asolution may be sterilized using filtration. However, sterilization bymeans of gamma radiation is preferred, since this can be carried outafter packaging, so that packaging under aseptic conditions isunnecessary. The loss of activity of the (lignolytic) polyphenoloxidase,such as laccase, due to gamma sterilization which can amount to up to50% may be compensated using a correspondingly higher initialconcentration.

The above-defined adhesive is preferably used as a medicament.Classification as a pharmaceutical preparation or a medical preparationis also possible depending on national law. These terms can beinterchanged in the context of the invention.

In the following, uses of the inventive kits and adhesives are describedin detail.

The finding of the present invention is that by means of bridgemolecules using (lignolytic) polyphenoloxidases, such as laccase,crosslinking may be achieved providing covalent bonds with polymers thathave at least one free amino group. Therefore, the present inventionrelates to the use of a kit or adhesive as defined above for the closureof wounds. It is, however, sufficient that the kit or adhesive comprisesonly the individual components (b) and (c) in order to cause theadhering of wounds or the like, as described further below. Thus, thepresent invention relates to the use of a kits comprising the followingindividual components:

(a) Bridge molecules, selected from the group consisting of monocyclicortho-dihydroxyaromates, monocyclic para-dihydroxyaromates, bicyclicmonohydroxyaromates, polycyclic monohydroxyaromates, bicyclicdihydroxyaromates, polycyclic dihydroxyaromates, bicyclictrihydroxyaromates, polycyclic trihydroxyaromates, and mixtures thereof,and

(b) Polyphenoloxidases, in particular lignolytic polyphenoloxidases,

wherein the individual components (components (a) and (b)), i.e. thebridge molecules with the polyphenoloxidases, especially lignolyticpoylphenoloxidases, are not in contact, for the manufacture of anadhesive for adhering wounds, in particular of hard and soft tissue, asdescribed in further detail below.

Thus, in the following, by kit or adhesive, a kit or adhesive is meantthat comprises the two individual components “bridge molecules” and“polyphenoloxidases” or the three individual components “polymers havingat least one free amino group,” “bridge molecules,” and“polyphenoloxidases,” wherein a kit or adhesive having the threeindividual components described here is preferred.

With respect to the individual components and their preferredembodiments, it is referred to the kit or adhesive defined above.

The kit and/or the adhesive can be used for adhering hard and softtissue. For example, connective tissue, skin, tendon, organ, bloodvessel, and/or nerve (soft tissue) and/or tooth and/or bone (hardtissue) can be adhered. Of course, different kinds of tissues can beadhered to each other, e.g. tendons and bones. A particularly preferredapplication is in adhering of wound edges or in adhering ruptures ofvarious organs, such as liver, kidney, or spleen. Treatment of surgicalwounds, e.g. after removal of tumors, is also possible with theinventive kit and/or adhesive. Further fields of application are: inplastic, reconstructive, and/or cosmetic surgery, in particular forpreventing the formation of scars induced during suturing of wounds.

Closure and sealing of liquid and air leakages, e.g. sealing of stitchchannel bleeding in the surgery of vessels, in arterial bypass surgeryor for sealing leakages of the lung in thoracic surgery.

An advantageous application of the kits and/or adhesives is theflexible, over a certain period of time resorbable sealing ofanastomases and patches on vessels or hollow organs.

The kit and/or adhesive is also suitable for fixation of drug deliverydevices and porous carrier structures or membranes for potential use inregenerative medicine (tissue engineering). Here it is of a specialadvantage that the adhesion can also be carried out with surfaces ofnon-biological origin, as long as they carry free amino groups. Freeamino groups can also be obtained on such surfaces of non-biologicalorigin according to methods known in the art, e.g. plasma methods(Schröder et al., Improved low-pressure microwave plasma assisted aminofunctionalization of polymers, Plasma processes and polymers, Weinheim,Deutschland, Weinheim: Wiley-VCH, 2005; Schroder et al., Plasma-InducedSurface Functionalization of Polymeric Biomaterials in Ammonia Plasma,Contrib. Plasma Phys. 41, 2001, 562-572; Meyer-Plath et al., Currenttrends in biomaterial surface functionalization—nitrogen-containingplasma assisted processes with enhanced selectivity, Vacuum 71, 2003,391-406).

The surfaces of e.g. implants or of drug delivery devices intended to beconnected with adjacent tissue using the kit and/or adhesive should havea content of at least 2% free amino groups. The term “surface” refersnot only to flat areas, but to boundary areas of a solid substrate inany form. Thus, substrates can also be beads or surfaces of complexshape.

Adhesion methods using the above-identified kits and/or adhesives usablein an aqueous environment are also an embodiment according to theinvention that leads to a strong, crosslinked resorbable connection.This is due, in particular, to a strong connection caused bycrosslinking via polymers carrying free amino acids and bridge moleculesinduced by (lignolytic) polyphenoloxidase, such as induced by laccase,as defined above.

The invention especially relates the method for adhering soft and hardtissue using a kit and/or adhesive as defined above. When adhering softand hard tissue, the adhering of bones to bones may be concerned. Heresmall bone fragments that due to their size cannot be fixed at all oronly unsatisfactorily using screws are fixed using the inventive kitand/or adhesive. Compared to metallic implants, a second surgery afterhealing of the fracture is unnecessary when using adhering by means ofthe inventive kit and/or adhesive. Furthermore, bones may be fixed tosoft tissue using the inventive kit and/or adhesive. In particular,cartilage transplants (e.g. from tissue cultures), e.g. for treatingarthritis, may be fixed sufficiently by means of the inventive kitand/or adhesive until growth onto a joint surface. Of particularadvantage is the fact that the kit and/or adhesive is successivelyresorbed. Furthermore, implants can be fixed by means of the inventivekit and/or adhesive to bones. The adhesive force of screws is ratherdecreased, especially in osteoportic bones. The adhesive force of screwsmay be supported by adhering the outer surface of bones on a great areawith the implant (e.g. a plate) by means of the inventive kits and/oradhesives, or screws may even totally be dispensed. Studies of cellvitality showed a good biocompatibility of the crosslinking product.

Accordingly, the invention comprises, in particular, also the use of theinventive kit, either having the two individual components “bridgemolecules” and “polyphenoloxidases” or especially preferred having thethree individual components “polymers having at least one free aminogroup,” “bridge molecules,” and “polyphenoloxidases” as defined above,for the manufacture of the inventive adhesive for the closure of wounds.By wounds, interruptions of the continuity of outer and inner bodysurfaces, such as e.g. incision wounds or fractures are meant.Accordingly, the invention, in particular, also comprises the use of theinventive kit for the manufacture of the adhesive for adhering of softand hard tissue as well as adhering of bones with implants (e.g. aplate), as described above.

A very strong adhesion is formed in various fields of application,which, however, can be resorbed during the course of the healingprocess, by the crosslinking by means of the inventive combination, inparticular by the bonding of peptide sequences containing the aminoacids relevant for the adhesive action using the enzyme systems(lignolytic) polyphenoloxidase, such as laccase. The initial healingphase in connection with good adhesive properties and the time-dependentslow resorption into the body during advanced healing processes are ofspecial advantage.

The adhesive or the kit can be used in bone fracture for fixing vesselprostheses, biodegradable implants, catheters, stents, and othermaterials.

Accordingly, the invention comprises the use of the inventive kits,either with the two individual components “bridge molecules” and“polyphenoloxidases” or especially preferred with the three individualcomponents “polymers having at least one free amino group,” “bridgemolecules,” and “polyphenoloxidases” as defined above for themanufacture of an adhesive for adhering bone fractures and/or for fixingvessel prostheses, biodegradable implants, catheters, stents, and othermaterials.

Compatibility of implanted material with the bone cells is offundamental importance for the adhering of bones as well as for therapid integration of implants, in particular where bones are concerned.This, of course, also refers to the used adhesive. Due to the rapidgrowth of osteoblasts and their subsequent ripening, the formation ofnew bone is promoted and the healing process is shortened. Therefore,implants are provided with microstructured surfaces. The inventive kitor adhesive maintains this structure and supports the formation of newbone. The amino acids lysine and proline are required for the productionof collagen, which is necessary for the healing of bones.

Preferably, (lysine-thyrosine)_(n) is used as a polymer for this fieldof application. When using (lysine-thyrosine)_(n) as a polymer, lysineis present at the adhesion site in high concentration. This results in apositive influence on the formation of bones. The formed extracellularmatrix is identified using integrines, such as α1β1 or α3β1 because ofthe RDG sequence (Arg-Gly-Asp). This induces a signal cascade thatcauses changes in the cytoskeleton in the cell and switches between thestages of proliferation and differentiation. The formed protein matrixfurther serves the incorporation of calcium salts.

This enables the application in complicated fractures and theincorporation of bone fragments. The advantages of the inventivecombination of stopping of bleeding and high adhesive force are of greatimportance especially in the surgery of complicated fractures.

In a further aspect, the invention relates to novel zeolite granulatessuitable for the stopping of bleeding that can be applied individuallyas well as used in addition to the inventive kit.

In particular, a rapid adhesion of the edges of wounds is achieved bythe novel zeolite granulates.

The novel zeolite granulates are zeolite granulates having at least 70%by weight of zeolite, in particular having a proportion ofclinoptilolite, chabasite, and mordenite, and a micropore volume havinga medium pore size of 0.3-0.5 nm. Preferably, these zeolite granulatesare free of hydrate, wherein this condition is preferably achieved afterdrying and precrushing by mild dehydration at temperatures below 200° C.In the mild dehydration, various temperature intervals below 200° C.are, in particular, used. Especially preferably, zeolite granulates areused that originate from the fraction having a corn band of 0.8-0.2 mm.

Regarding the purity of these zeolite granulates, all requirementsaccording to US Pharmacorp. or Europ. Pharmacorp. are to be satisfied.

It has been found that certain factors involved in blood coagulation areselectively enriched in the micropore volume of these zeolitegranulates. This leads to an activation of the physiological bloodcoagulation that causes the stopping of bleeding which is significantlysuperior over conventional haemostasis. In this connection, it is ofspecial advantage that no toxic compounds are released to the wound. Inthe described inventive preparation, a zeolite granulate having strongelectrostatic fields in the crystal lattice is obtained that can evenstop strong bleeding within 1 min. Surprisingly, it has been found thatthe inventive zeolite granulates have a capillary force action that isfive to ten times superior over products on the basis of microporoussynthetic lithium silicates (EP 1 176 991 A1, WO 00/69480, U.S. Pat. No.6,833,486 B1), e.g. CERDAC, available on the market. Due to the partialdehydration, no exothermal reaction occurs that may cause tissue damageby burning.

By means of the described inventive preparation, natural zeolites cannow be provided for the first time in a cost-effective, direct way asmeans for stopping bleeding and means for the treatment of wounds.Accordingly, the present invention also comprises the inventive zeolitegranulates as a medical product or as a medicament and the use of theinventive zeolite granulates for the manufacture of a medical productand/or a medicament for stopping of bleeding. As in the case of the kit,the classification as a pharmaceutical preparation or as a medicalproduct, depending on national law, is possible. These terms are used inthe context of the description of the invention synonymously.

As already mentioned above, it has been found that by means of theinventive zeolite granulates not only stopping of bleeding is achieved,but also a rapid adhesion of the wound edges. After the adhesion of thewound edges, the mineral material may be completely removed, forexample, by detaching the scab-like wound coverage and extracting theremains. In this embodiment of the invention, it is possible to applythe zeolites in a covering, e.g. on the basis of cellulose or on atextile basis or on the basis of a collagen non-woven material. Thus,the subsequent complete removal is possible without any problems.

The wound edges are adhered to each other by activating the fibrinsystem. Accordingly, the invention also comprises the use of theinventive zeolite granulate for the manufacture of the inventiveadhesive for adhering wounds as well as for sealing anastomases andpatches to vessels or hollow organs, respectively.

The tear resistance of the wound closure is, however, lower than inconventional fibrin adhesives.

In order to increase the tear resistance, it is useful to use theinventive zeolite granulates in the above-described kit or adhesive. Bythis combination, especially effective haemostatic wound adhesives canbe prepared for treatment of wounds, sealing of wounds, and stopping ofbleeding. Accordingly, the present invention relates in a furtherembodiment to a kit, comprising the following individual components:

(a) Polymers having at least one free amino group,

(b) Bridge molecules, selected from the group consisting of monocyclicortho-dihydroxyaromates, monocyclic para-dihydroxyaromates, bicyclicmonohydroxyaromates, polycyclic monohydroxyaromates, bicyclicdihydroxyaromates, polycyclic dihydroxyaromates, bicyclictrihydroxyaromates, polycyclic trihydroxyaromates, and mixtures thereof,

(c) Polyphenoloxidases, in particular lignolytic polyphenoloxidases, and

(d) Minerals for stopping bleeding, in particular zeolite granulates asdescribed above,

wherein both individual components (b), (c), and (d), i.e. the bridgemolecules with the lignolytic polyphenoloxidases and the minerals forstopping bleeding, are not in contact.

Regarding the individual components and their preferred embodiments, itis referred to the description above. Accordingly, the kit is based, inparticular, on natural zeolite fine granulates and on polyphenoloxidases(in particular, laccase) by which the poly or oligopeptides,respectively, can be crosslinked.

Here it is preferred that the natural zeolite fine granulates are not incontact with the other individual components of the kit, i.e. it ispreferred that the zeolite is packed separately and is used separately.Because of the high capillary force, the natural zeolite fine granulatescause a rapid closure of the wound by concentration of coagulationfactors. Subsequently, the zeolites may be removed without a problem. Aclear surgical area free of blood is maintained. It is furtheradvantageous that possibilities for corrections, e.g. before adding thenon-crosslinked polymers, if desired, are still given.

When the individual components (a) to (c) of the kit are initiallyprovisionally applied to the closed wound, a wound closure is formedunder development of covalent bonds that is, on the one hand, durableand loadable, but, on the other hand, also resorbable.

Of course, other means for stopping bleeding than the inventive zeolitegranulates that make it possible to strongly reduce the loss of bloodand, thus, removal of the (lignolytic) polyphenoloxidases, in particularlaccase, at the application site may be considered. It is also possibleafter stopping bleeding using the natural zeolite fine granulates to useother tissue adhesives. However, the procedure described above—firstlystopping bleeding and closing the wound using natural zeolite finegranulates and subsequently durable adhesion involving thepolyphenoloxidase, such as laccase—shows advantages and is superior overcompeting methods also under the viewpoint of costs. The inventivehaemostatic kit for the treatment of wounds, sealing of wounds, as wellas for adhering hard and soft tissue and implants is, furthermore,significantly easier to handle. The haemostatic adhesives canadvantageously be used in plastic, reconstructive, and/or cosmeticsurgery, in particular for avoiding the formation of scars inducedduring suturing of wounds, closure and sealing of liquid and airleakages, e.g. sealing of stitch channel bleedings in vessel surgery, inarterial bypass surgery, or for the sealing of lung leakages in thoracicsurgery.

The inventive kit is an alternative to electrocoagulation as well as toclosing of wounds using needle and thread.

Accordingly, the present invention also comprises the use of theinventive kit, comprising the individual components (b) to (d) or, morepreferably, (a) to (d) for the manufacture of an adhesive for theclosure of wounds. Furthermore, the invention in particular alsocomprises the use of the inventive kit, comprising the individualcomponents (b) to (d) or, more preferably, (a) to (d) for themanufacture of an adhesive for adhering hard and soft tissue as well asadhering bones with implants (e.g. a plate) as described above.

Furthermore, the present invention also relates to an adhesivecomprising components (b) to (d) or, more preferably, (a) to (d), asdescribed as above, wherein the components (b) to (c) or (a) to (c),respectively, preferably all individual components (b) to (d) or (a) to(d), respectively, are in contact.

The high capillary force of the natural zeolite fine granulates is alsoof special importance for use in the treatment of wounds, in particularfor the treatment of difficult-to-heal wounds and chronic wounds.Especially applicable is the use of the natural zeolite fine granulatesin wounds having an exsudation, i.e. leaking of blood components from aninfected wound. Exsudation is often an indication of an infection in aninjury. The microporous structure of the zeolite granulates obtainedaccording to the invention enables the absorption of wound exsudate thatis released in “wetting” wounds in excess. Simultaneously, theabsorption of excess exsudates provides a wet atmosphere over the wound,which is of importance for wound healing.

Further inventive uses are: as a means for first aid after accidents andfor sanitation of infected wounds.

Completely surprisingly, it was found that zeolites having a pore widthof 0.3-0.5 nm can be mixed with a biomass from aquatic organisms, asdefined further below, in particular in a ratio of 1:10 to 10:1 withoutloss of the stopping effect on the bleeding or of capillary force.

Accordingly, the use of the novel zeolite granulates can be extended inthat these materials are complemented with biomass. Accordingly, thepresent invention also comprises the inventive zeolite granulates, whichadditionally contain biomass, in particular in a ratio of zeolite tobiomass of 1:10 to 10:1. Preferably selected according to the inventionare microalgae having antibacterial activity, the biomass thereof islyophilized and milled and/or processed according to the technicalteaching of Lukowski et al.: Pharmaceutically or cosmetically activeagent obtained from lipid-containing marine organisms, U.S. Ser. No.10/507,061.

By mixing the natural zeolite fine granulates with biomass containingantimicrobial agents and being processed in that manner, it is possibleto provide a combination that ensures the absorption of excess woundexsudate, keeps the wound wet, prevents infection of the wound, isnon-inflammatory and immunostimulating, and promotes cell proliferation.Preferably selected in the preparation according to U.S. Ser. No.10/507,061 are microalgae having a lipid content of >20% having lots ofunsaturated fatty acids and/or having a high content ofpoly-N-acetyl-glucosamine. Thus, the antibacterial activity of the fattyacids can advantageously be used for this object. Polysaccharides,advantageously from poly-N-acetyl-glucosamines or chitosane have a greatinner surface. Due to the absorption onto this great surface, asynergistic effect regarding the concentration of coagulation factors aswell as the absorption of wound secretion and bacterial toxins results.Furthermore, these polysaccharides have immunostimulating action.According to a preferred embodiment of the invention, lyophilizedbiomass of the green algae chlamydomonas and/or the microalgae spirulinaand/or anabaena are used. By using the zeolites with or without biomasson a bleeding wound, a scab-like covering is formed by activating thefibrin system, which reliably protects the wound from bacterialinfection. This covering is repelled in the course of wound healing.

In a suitable embodiment, the fine granulates from natural zeolites andmicroalgae biomass can also be used for treating highly infected,wetting wounds.

In various fields of application, the kit and/or adhesive can containthe inventive combination of zeolite granulate and biomass in allembodiments described in detail above.

The features of the invention can be taken not only from the claims, butalso from the description, wherein the individual features on their ownor in the form of combinations of more than one feature, respectively,are advantageous, patentable embodiments for which patent protection isrequested in this application. The steps according to the invention canbe used individually.

The invention will be described in more detail according to workingexamples without being limited to these examples.

EXAMPLES Example 1 Preparation of the Natural Zeolite Fine Granulates

Selectively decomposed minerals having 70% zeolite content, preferablyclinoptilolite, chabasite, and mordenite, are obtained after drying andprecrushing with a corn band of 0.8 to 0.2 mm. With respect to purity,the requirements according to US Pharmacorp. or Europ. Pharmacorp. areto be satisfied. A partial dehydration while maintaining the crystallattice structure is obtained by the mild thermal treatment in selectedtemperature intervals at temperatures <200° C.

Example 2 Testing the Micropore Volume and the Grain Size Distribution

The products obtained according to Example 1 exhibit 25-50% microporevolume (relative to the total volume) and a medium nanopore diameter inthe range of 0.3-0.5 nm.

The nanopore size and the structure of the fine granulates enable amaximized blood absorption and a rapid penetration of the fine granulateresorbent.

The capillary force action is by five to ten times higher than in thecommercial product CERDAC tested for comparative purposes.

Example 3 Testing the Fine Granulates for their Suitability for StoppingBleeding

Method:

Two fine granulates, obtained according to Example 1, are tested.

The tests were carried out using rat liver as a prototype for an organstrongly supplied with blood. For this reason, rats were narcotized.After opening the abdominal area, a long cut of about 1 cm in length wasmade with a scalpel. The bleeding was filmed. The time period untilstopping of the bleeding was measured.

Result:

The application of the inventive granulates leads to the stopping of thebleeding within 1 min. (control without treatment up to 8 min.). Ascab-like covering is formed protecting the wound.

Example 4 In-Vitro Testing of the Fine Granulates for their Suitabilityfor Stopping of Bleeding

The following results were obtained in tests using various blood plasma:

a) Normal plasma (with pre-incubation of 2 min.)

Normal plasma Granulate 1 Granulate 2 Recalcification time 115 s 60 s 60s PTT after 3-min. — 15 s Immediate clot incubation Clot = Coagulation

b) Deficient Plasma

Granulate 1 Granulate 2 F - VII MPI Clot after 5 min. Clot after 210 sThere was no coagulation without granulate.

c) Heparine plasma (with 2 and 3 min. preincubation)

Without Granulate 1 Granulate 2 granulate 2 min. 3 min. 2 min. 3 min. 1.68 s 46 s 43 s 65 s 28 s 2. 45 s 38 s 17 s 37 s 10 s

The results show that the endogenous coagulation system is activated,wherein the deficiency factor F-VII and tissue factor lead to increasedtrombine and fibrin formation. A relative increase of the fibrinconcentration due to the mineral wound coverage according to Example 1is also obtained.

Example 5 Testing of Extracts from Microalgae in View of theirAntibacterial Efficiency

Method:

Various staphylococcus strains were uniformly distributed by means of aWhitley Automatic Spiral Plater on the agarplate (Willer-Hinton-II-Agarready-to-use plates by Becton Dickinson).

An amount of extract solution corresponding to 2 mg extract is droppedonto acetate tissue as used for the manufacture of a wound coverage andis allowed to dry.

Lyophilized biomasses of the green algae chlamydomonas, of themicroalgae spirulina (Blue Biotech BUsum) and Anabaena (straincollection by the Department of Pharmacy at the Ernst-Moritz-ArndtUniversity of Greifswald) were used for the manufacture of the extracts.The extractor DIONEX ASE 200 was used for the extraction with n-hexane.The extract solution was separated from the solvent using a rotaryevaporator.

Result:

Seventy to one hundred twenty seeds/cm² were counted in the control. Nogerms of staphylococcus aureus strains resistant to methicillin (MRSA)that may cause wound infections were found beneath the wound coverage.Other straphylococca showed a reduced germ growth of 10% to 20% of theinitial germ number.

Example 6 Manufacture of Fine Granulates from Natural Zeolites and theBiomass of Microalgae

Microalgae biomasses having a lipid content of >20% can be admixed tothe mineral fine granulates obtained according to Example 1 in a ratioof 1:10 to 10:1. Thus, infection of wounds can be prevented.

Example 7 Testing of the Fine Granulates for Their Suitability forStopping Bleeding and for Wound Closure

a) Method:

Two fine granulates, obtained according to Example 1, were tested.

The tests were carried out using rat liver as a prototype for an organstrongly supplied with blood. For this reason, the rats were narcotized.A long cut of about 1 cm in length was made with a scalpel after openingof the abdominal area. The bleeding was filmed. The time period untilstopping of the bleeding was measured. The wound closure was ratedvisually.

Result:

The application of the inventive granulate leads to a stopping of thebleeding within 1 min. (control up to 8 min.). A scab like covering isformed protecting the wound.

b) The covering was removed with a scalpel after 1 min. in furthertests.

Result:

A wound having a superficially closed surface that is no longer bleedingand having a visually rated good wound closure is obtained. The woundclosure in case of fine granulates on mineral material/algae biomassbasis is superior over the use of preparations that are exclusivelyprepared on a mineral basis.

c) The fine granulates were separated from the wound in further testsusing a cellulose patch.

Result:

Under these conditions, stopping of bleeding within less than 1 min. isobtained. A clear surgical area is obtained so that a strong adhesion ofthe wound using the tissue adhesive according to the invention can becarried out without any problem.

Example 8 Manufacture of the Adhesive Prepolymers and Carrying Out ofthe Adhesive Reaction for Adhesion of Hard and Soft Tissue

The developed adhesive results in mixture of the individual componentsoligopeptide 1 (amino acid sequence consisting of dipeptide repeatingunits of tyrosine and lysine, n=5 or n=10), bridge molecule 2(2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide) and the enzyme laccase(Scheme 5). The tests were carried out in phosphate buffer.

The adhesive action is based on the enzymatic reaction of the laccasewith the bridge molecule that is being connected with the oligopeptide.

The following mixing ratios were employed:

[Tyr-Lys]_(n), n=4-35, 8.5 mM;2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide 12.5 mM; enzyme: 0.32 U (156nmol ml⁻¹ min⁻¹). The solvent content of methanol was 10% (v/v).

Example 9 Testing of the Firmness of the Adhesion Seam

For testing the firmness of the adhesion seam in the pulling test, thetissue parts to be tested had to be fixed in advance. Porcine softtissue was fixed with cyanacrylate adhesive on sample carriers made ofpolymethyl methacrylate (PMMA) or bone plates of bone of bovine originwere clamped in mechanical clamps. Subsequently, the adhesive mixture tobe tested (cf: Example 6) was applied to the tissue. When adhering softtissue, the firmness of the adhesion seam was determined after 10minutes under pulling stress in the measuring apparatus Zwick BZ2.5/TN1S(scan rate: 10 mm/min). The mechanical testing of the adhesion seam whenadhering hard tissue was carried out using pulling shear stress with ascan rate of 5 mm/min.

Result: Two times the firmness is obtained in comparison to the testedcommercially available fibrin adhesives.

Example 10 Diffusion Inhibition Test Using a Collagen Non-Woven MaterialTreated According to the Invention in Comparison to a Non-Woven MaterialTreated with a Conventional Antibiotic

Method:

The agar diffusion test according to Burkhardt (Burkhardt, F. (Hrsg.)Mikrobiologische Diagnostik. Georg-Thieme Verlag Stuttgart, New York1992, p. 724) was carried out. Mueller-Hinton II-Agar in Stacker Petridishes (Becton Dickinson Microbiology Systems, Cockeysville, USA) wereemployed in the test for 15. The strain S. aureus ATCC 6538 was selectedas test strain. The initial seed of this test strain was chosen suchthat after 1620 hours of incubation, closely standing, but notconfluent, single colonies had developed. After drying of the seededculture media, the treated collagen non-woven material was applied tothe agar surface. After 18+2 hours incubation at 36° C., the inhibitionsphere was measured.

Results:

Inhibition sphere against S. aureus ATCC 6538 Non-woven materialTreatment Gentamycine treated acc. to the invention 15% 30 20% 24 36 30%38 40% 38

The non-woven material according to the invention has the followingadvantages: improved antimicrobial activity, also againstmulti-resistant germs, very good haemostatic action, and good adhesionon the wound and easy application.

Example 11 Testing of Surfaces Treated with Polyphenols According to theInvention Regarding Antibacterial Efficiency

Method:

Various staphylococcus strains were uniformly distributed on the agarplate (Mueller-Hinton-II-Agar ready-to-use plates of Becton Dickinson)by means of a Whitley Automatic Spiral Plater in linear modus.

Polymer biomaterials (1 cm² each) were functionalized according toContrib. Plasma Phys. 41, 2001, 562-572 in ammonik plasma. Subsequently,reaction with 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide (12.5 mM) underthe influence of the laccase (0.32 U (156 nmol ml⁻¹ min⁻¹) was carriedout. The solvent content of methanol was 10% (v/v).

After drying, the plates were put onto the agar.

Result:

One hundred twenty germs/cm² were counted in the control. Under thecoated surfaces no germs could be detected.

Example 12 Use of the Combination According to the Invention forTreating Bleeding Wounds

Method:

Rats were narcotized as in Example 7, the abdominal area was opened anda cut was made into the liver. The bleeding was stopped as in Example7c, the crust in formation was removed. Subsequently, the cut was fixedwith wound adhesive.

For this purpose, the oligopeptides described in Example 6 wereincorporated in PBS (phosphate buffered saline, 2.7 M NaCl, 54 mM KCl,87 mM Na₂HPO₄, 30 mM KH₂PO₄, pH 7.4) and laccase was added (component1). 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide was dissolved in PBS(component 2). Different ratios ofoligopeptide/2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide/laccase weretested, e.g. 1/1/0,1; 1/2/0,5; 1/10/1). A minimum amount of solvent wasselected in order to have a solution of the components of maximumconcentration. The components were put into a two-compartment syringehaving a mixing extruder of the type Mixpac (Mixpac Systems, Rotkreuz,Switzerland) and were applied to the wound fissure.

Result: A good wound closure was achieved with haemostatics. Theadhesive can be put directly into the resulting narrow fissure. Theresult is a very strong adhesion of the wound edges.

Example 13 Adhering of Collagen

Method:

Collagen foil (DOT GmbH) was fixed on a flat carrier.

The collagen foil was divided into 8 segments (or in two rows) and theadhesive was mixed directly on the foil (by using the surface tension).The adhesive was dissolved in phosphate citrate buffer.

Different concentrations of bridge molecules (laccase substrate) weretested. The adhesive protein had an MW of about 4,000 D.

Subsequently, small pieces of collagen foil were applied, pressed softlyand were allowed to dry.

Result:

When adhering collagen, a strong bond was achieved.

Adhesion also results without the addition of adhesive proteins.However, the adhesive force was increased by the adhesive proteins.

Example 14 Mixing of a Collagen Solution with the Adhesive

A significant structuring can be detected under the microscope whenmixing the collagen solution with the adhesive. The dissolved andincidentally ordered collagen molecules were made parallel while formingfibers.

Example 15 Adhering Collagen and Polystyrene

Method:

A drop of citrate phosphate buffer (20 μl, control) or adhesive solution(buffer, laccase, laccase substrate) were applied onto the surface of a60 mm TCPS cell culture vessel and a small piece of collagen foil (8×4mm) was applied, respectively, pressed smoothly and was then allowed todry.

Result:

Despite the smooth and inert surface, an adhesion was achieved. Thecollagen was durably bonded to the carrier.

Example 16 Cell Adhesion onto the Surfaces Loaded with Collagen

Method:

Cell adhesion was tested using a fibroblast cell line (FL cells).

Cell adhesion occurred on a 60 mm TCPS cell culture vessel that wascoated with collagen as in Example 15. After coloring the cells withcrystal violet, evaluation was done with the CellExplorer.

Result:

The FL cells were bonded more strongly (cf: FIG. 1).

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

The invention claimed is:
 1. A granulate comprising a zeolite, wherein acontent of said zeolite in said granulate is at least 70% by weight,said zeolite having a medium pore size of 0.3 nm to 0.5 nm, and whereinsaid granulate is at least partially dehydrated wherein said granulatefurther comprises a biomass made from aquatic organisms, and whereinsaid biomass comprises a lipid content of greater than 20%.
 2. Thegranulate of claim 1, wherein the granulate has a medium grain size of0.2 mm to 0.8 mm.
 3. The granulate of claim 1, wherein said aquaticorganisms have antibacterial activity or havepoly-N-acetyl-glucosamines.
 4. The granulate of claim 1, wherein saidbiomass comprises a lyophilized biomass selected from the groupconsisting of a green algae chlamydomonas, a microalgae spirulina, andan anabaena.
 5. The granulate of claim 1, wherein said biomass and saidzeolite are mixed in a ratio of from 1:10 to 10:1.
 6. The granulate ofclaim 1, wherein said granulate and said biomass are contained in anencasement.
 7. A medicament comprising said granulate of claim
 1. 8. Amethod of closing a wound comprising applying said granulate of claim 1to a wound.
 9. The method of claim 8, wherein said wound is ananastomosis of a vessel or a hollow organ.
 10. The granulate of claim 1,wherein said zeolite is selected from the group consisting ofclinoptilolite, chabasite, mordenite and mixtures thereof.
 11. Agranulate comprising a zeolite, wherein a content of said zeolite insaid granulate is at least 70% by weight, said zeolite having a mediumpore size of 0.3 nm to 0.5 nm, and wherein said granulate is at leastpartially dehydrated wherein said granulate further comprises a biomassmade from aquatic organisms, and wherein said biomass comprises alyophilized biomass selected from the group consisting of a green algaechlamydomonas, a microalgae spirulina, and an anabaena.
 12. Thegranulate of claim 11, wherein the granulate has a medium grain size of0.2 mm to 0.8 mm.
 13. The granulate of claim 11, wherein said aquaticorganisms have antibacterial activity or havepoly-N-acetyl-glucosamines.
 14. The granulate of claim 11, wherein saidbiomass and said zeolite are mixed in a ratio of from 1:10 to 10:1. 15.The granulate of claim 11, wherein said granulate and said biomass arecontained in an encasement.